Multi-arm polypeptide-poly(ethylene glycol) block copolymers as drug delivery vehicles

ABSTRACT

The invention provides a multi-arm block copolymer for use in delivering a variety of bioactive agents. The copolymer of the invention contains a central core from which extend multiple (3 or more) copolymer arms. Each copolymer arm possesses an inner polypeptide segment and an outer hydrophilic polymer segment. Thus, the overall structure of the copolymer comprises an inner core region that includes the central core and the inner polypeptide segment, while the outer core region is hydrophilic in nature. The multi-arm copolymer of the invention is particularly useful for delivery of biologically active agents that can be entrapped within the inner core region.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/746,567, filed Dec. 24, 2003, which claims the benefit of U.S.Provisional Patent Application Ser. No. 60/437,372 filed on Dec. 30,2002, the disclosures of each are hereby incorporated herein byreference in their entireties.

FIELD OF THE INVENTION

The invention relates to multi-arm copolymers and methods of making andusing such copolymers as, for example, drug delivery vehicles.

BACKGROUND OF THE INVENTION

The development of effective-drug delivery vehicles continues to presenta challenge for drug manufacturers. Many bioactive agents, whiledisplaying potent in vivo activity, are hampered by drawbacks such as invivo degradation, rapid elimination from the body, low aqueoussolubility, and systemic toxicity. Several approaches have beensuggested to overcome these drawbacks. Such approaches include, forexample, co-administering a bioactive agent with a surfactant, providingthe bioactive agent in a liposomal formulation, providing targeting to aspecific tissue by employing an antibody, and formulating the bioactiveagent within a micelle. Each approach, however, does not fully addressthe problems encountered with the specific active agent and/or generatesadditional challenges.

Pharmaceutical grade surfactants, such as Tween 80 or Cremophor®, havebeen widely used in formulations to compensate for the low aqueoussolubility of hydrophobic drugs. These surfactants solubilizehydrophobic drugs by forming micellar structures in aqueous media.Unfortunately, these surfactants have been associated with severeallergic reactions and hypersensitivity when administered to patients(Kris et al. (1986) Cancer Treatment REP 70:5. In addition, micellardrug carriers often disintegrate upon administration to a patientbecause the concentration of the component forming the micelle fallsbelow its critical micelle concentration (CMC). Once the micelledisintegrates, there is a rapid and uncontrolled release of the drug,thereby often rendering this approach to drug delivery as impracticable.

Liposomal formulations are made up of phospholipids that form liposomes.Upon administration to a patient, the liposomes are taken up bymacrophages of the reticulo-endothelial system (“RES”). High levels areoften found in the liver and spleen, even when the liposomes aremodified to possess “stealth” characteristics by coating them withpoly(ethylene glycol) (“PEG”). Even PEG-coated “stealth” liposomes,however, possess undesirable side effects. In particular, suchPEG-coated liposomes are known to indiscriminately move from the bloodvessels into tissues, a process known as “extravasation.” As a result,higher doses of liposome-encapsulated drug must be administered toachieve a desired therapeutic effect.

Targeted delivery approaches, e.g., using antibodies to deliver drugssuch as anticancer agents, have been employed for localized treatment ofdiseases such as cancer. Unfortunately, the receptors being targeting onthe tumor cells are often present on healthy cells as well. Thus,antibody-targeting approaches often lack the specificity or selectivitynecessary for providing an optimized method for delivering a bioactiveagent.

Still other approaches have been suggested for delivering drugs. Forexample, water-soluble polymers such as poly(ethylene glycol) have beencovalently attached to drugs to form polymer-drug conjugates. Suchconjugates often possess improved water solubility, enhanced in vivostability, and an improved therapeutic index in comparison to theunconjugated or native drug. Unfortunately, monofunctional PEGs, such asmonomethoxy-PEG, can carry only one drug molecule per polymer chain,thereby lacking the high drug-carrying capability often sought indelivering bioactive agents.

Thus, there remains a need in the art for improved methods fordelivering both hydrophilic and hydrophobic drugs in a therapeuticallyeffective manner. That is to say, there is a need for compositions andmethods of drug delivery that are flexible enough to be useful fordelivering not only water-insoluble drugs, but are adaptable for use indelivering hydrophilic and charge-bearing bioactive agents as well. Thepresent invention seeks to solve these and other needs in the art.

SUMMARY OF THE INVENTION

The invention provides a unimolecular multi-arm block copolymercomprising a central core molecule providing at least three attachmentsites available for covalent attachment, and a copolymer arm covalentlyattached to each of the attachment sites of the central core molecule.Each copolymer arm comprises an inner polypeptide segment covalentlyattached to the central core molecule and an outer hydrophilic polymersegment covalently attached to the polypeptide segment. The central coremolecule and the polypeptide segments taken together define an innercore region and the hydrophilic polymer segments define a hydrophilicouter region. Biologically active agents can be entrapped within thecopolymer structure, preferably within the inner core region of theblock copolymer. Depending on the structure and properties of thebiologically active agent and the block copolymer, the entrapment canresult from various bonding or attraction forces, such as covalentbonding or attraction based on shared hydrophobicity or chargeattraction.

The central core molecule is preferably a polyamine. An exemplarypolyamine comprises a residue of a polyol core attached through etherlinkages to small molecular weight hydrophilic oligomers, such as PEGoligomers, each oligomer having a terminal amine group.

The polypeptide segment preferably comprises hydrophobic amino acidresidues, amino acid residues bearing an electric charge, or amino acidresidues having pendant functional groups suitable for covalentattachment to drug molecules. Exemplary amino acid residues includeresidues of glycine, alanine, valine, leucine, isoleucine, methionine,proline, phenylalanine, tryptophan, serine, threonine, asparagine,glutamine, tyrosine, cysteine, lysine, arginine, histidine, asparticacid, glutamic acid, and combinations thereof.

The hydrophilic polymer segment is preferably a poly(ethylene glycol)(“PEG”), optionally terminated with a capping group, such as alkoxy, ora functional group, such as hydroxyl, active ester, active carbonate,acetal, aldehyde, aldehyde hydrate, alkyl or aryl sulfonate, halide,disulfide, alkenyl, acrylate, methacrylate, acrylamide, active sulfone,amine, hydrazide, thiol, carboxylic acid, isocyanate, isothiocyanate,maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide,epoxide, glyoxal, dione, mesylate, tosylate, or tresylate. Thefunctional group can be used to covalently attach a drug molecule, ifdesired. In one particular embodiment, the PEG polymer segments bear atargeting moiety that can direct the copolymer to particular siteswithin the body for targeted release of the physically entrapped drug.Examples of targeting moieties include proteins, antibodies, antibodyfragments, peptides, carbohydrates, lipids, oligonucleotides, DNA, RNA,or small molecules having a molecular weight less than 2,000 Daltons.

When a hydrophobic polypeptide segment is employed, the block copolymerforms a unimolecular micelle structure wherein the central core and thepolypeptide segment define a hydrophobic core region. By entrapping oneor more hydrophobic biologically active agents within this hydrophobiccore region, the hydrophobic agent(s) become solubilized. In this way,it is possible to provide a means for administering previouslyunadministerable agents possessing hydrophobic-limiting solubility.Thus, improved delivery of a hydrophobic agent can be achieved byadministering a pharmaceutical composition containing a multi-arm blockcopolymer of the invention having a drug entrapped within itshydrophobic core region.

The invention further encompasses, among other things, pharmaceuticalcompositions comprising such block copolymers, methods of making thecopolymers, and methods of using the block copolymers as, for example,drug delivery vehicles.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus described the invention in general terms, reference will nowbe made to the accompanying figures, wherein:

FIG. 1 is an illustration of the general structure of the unimolecularmulti-arm block copolymer of the invention;

FIG. 2 is an illustration of the structure of an embodiment of theunimolecular multi-arm block copolymer having a charged inner coreregion;

FIG. 3 is an illustration of the structure of an embodiment of theunimolecular multi-arm block copolymer having a biologically activeagent covalently attached to the polypeptide segment of the copolymer;and

FIG. 4 illustrates the change in absorbance over time for severalconcentrations of cis-diamminodichloro platinum in a solution containingan 8-arm unimolecular block copolymer of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully. This inventionmay, however, be embodied in many different forms and should not beconstrued as limited to the embodiments set forth herein; rather, theseembodiments are provided so that this disclosure will be thorough andcomplete, and will fully convey the scope of the invention to thoseskilled in the art.

I. DEFINITIONS

It must be noted that, as used in this specification and the claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “adrug” includes a single drug as well as two or more of the same ordifferent drugs, reference to a “unimolecular multi-arm block copolymer”includes a single unimolecular multi-arm block copolymer as well as twoor more of the same of different unimolecular multi-arm blockcopolymers, reference to an “excipient” includes a single excipient aswell as two or more of the same or different excipients, and the like.

The terms “functional group,” “active moiety.” “activating group,”“reactive site,” “chemically reactive group,” and “chemically reactivemoiety” are used in the art and herein to refer to distinct, definableportions or units of a molecule. The terms are somewhat synonymous inthe chemical arts and are used herein to indicate the portions ofmolecules that perform some function or activity and are reactive withother molecules. The term “active,” when used in conjunction withfunctional groups, is intended to include those functional groups thatreact readily with electrophilic or nucleophilic groups on othermolecules, in contrast to those groups that require strong catalysts orhighly impractical reaction conditions in order to react (i.e.,“nonreactive” or “inert” groups). For example, as would be understood inthe art, the term “active ester” includes those esters that reactreadily with nucleophilic groups such as amines. Exemplary active estersinclude N-hydroxysuccinimidyl esters or 1-benzotriazolyl esters.Typically, an active ester will react with an amine in aqueous medium ina matter of minutes, whereas certain esters, such as methyl or ethylesters, require a strong catalyst in order to react with a nucleophilicgroup. As used herein, the term “functional group” includes protectedfunctional groups.

The term “protected functional group” refers' to the presence of aprotecting group or moiety that prevents reaction of the chemicallyreactive functional group under certain reaction conditions. Theprotecting group will vary depending on the type of chemically reactivegroup being protected and the reaction conditions employed. For example,if the chemically reactive group is an amine or a hydrazide, theprotecting group can be selected from the group of tert-butyloxycarbonyl(t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). If the chemicallyreactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also be usedin the invention, see for example, Greene, T. W., et al., PROTECTIVEGROUPS IN ORGANIC SYNTHESIS, 3rd ed., John Wiley & Sons, New York, N.Y.(1999).

The terms “linkage” and “linker” are used herein to refer to an atom,group of atoms, or bond(s) that are normally formed as the result of achemical reaction. A linker of the invention typically links adjacentmoieties, such as two polymer segments, via one or more covalent bonds.Hydrolytically stable linkages are linkages that are substantiallystable in water and do not react to any significant degree with water atuseful pHs, e.g., physiological pH, for an extended period of time,perhaps even indefinitely. A hydrolytically unstable or degradablelinkage is a linkage that is degradable in water or in aqueoussolutions, including for example, blood, plasma or other physiologicalfluid. Enzymatically unstable or degradable linkages encompass thoselinkages that can be degraded by one or more enzymes.

The term “alkyl” refers to hydrocarbon chains typically ranging fromabout 1 to about 12 carbon atoms in length, preferably from about 1 toabout 6 atoms, and includes straight and branched chains. Thehydrocarbon chains may be saturated or unsaturated.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or spiro cyclic compounds, preferablycomprising 3 to about 12 carbon atoms, more preferably 3 to about 8.

The term “substituted alkyl” or “substituted cycloalkyl” refers to analkyl or cycloalkyl group substituted with one or more non-interferingsubstituents, such as, but not limited to, C₃₋₈ cycloalkyl, e.g.,cyclopropyl, cyclobutyl, and the like; acetylene; cyano; alkoxy, e.g.,methoxy, ethoxy, and the like; lower alkanoyloxy, e.g., acetoxy;hydroxy; carboxyl; amino; lower alkylamino, e.g., methylamino; ketone;halo, e.g. chloro or bromo; phenyl; substituted phenyl, and the like.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁₋₆ alkyl (e.g., methoxy or ethoxy).

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Multiple aryl rings may be fused, as in naphthyl or unfused, asin biphenyl. Aryl rings may also be fused or unfused with one or morecyclic hydrocarbon, heteroaryl, or heterocyclic rings.

“Substituted aryl” is aryl having one or more noninterfering groups assubstituents. For substitutions on a phenyl ring, the substituents maybe in any orientation (i.e., ortho, meta or para).

“Heteroaryl” is an aryl group containing from one to four heteroatoms,preferably sulfur, oxygen, nitrogen, or a combination thereof, whichheteroaryl group is optionally substituted at carbon or nitrogen atom(s)with C₁₋₆ alkyl, —CF₃, phenyl, benzyl, or thienyl, or a carbon atom inthe heteroaryl group together with an oxygen atom form a carbonyl group,or which heteroaryl group is optionally fused with a phenyl ring.Heteroaryl rings may also be fused with one or more cyclic hydrocarbon,heterocyclic, aryl, or heteroaryl rings. Heteroaryl includes, but is notlimited to, 5-membered heteroaryls having one hetero atom (e.g.,thiophenes, pyrroles, furans); 5-membered heteroaryls having twoheteroatoms in 1, 2 or 1,3 positions (e.g., oxazoles, pyrazoles,imidazoles, thiazoles, purines); 5-membered heteroaryls having threeheteroatoms (e.g., triazoles, thiadiazoles); 5-membered heteroarylshaving 3 heteroatoms; 6-membered heteroaryls with one heteroatom (e.g.,pyridine, quinoline, isoquinoline, phenanthrine,5,6-cycloheptenopyridine); 6-membered heteroaryls with two heteroatoms(e.g., pyridazines, cinnolines, phthalazines, pyrazines, pyrimidines,quinazolines); 6-membered heteroaryls with three heteroatoms (e.g.,1,3,5-triazine); and 6-membered heteroaryls with four heteroatoms.

“Substituted heteroaryl” is heteroaryl having one or more noninterferinggroups as substituents.

“Heterocycle” or “heterocyclic” means one or more rings of 5-12 atoms,preferably 5-7 atoms, with or without unsaturation or aromatic characterand at least one ring atom that is not carbon. Preferred heteroatomsinclude sulfur, oxygen, and nitrogen. Multiple rings may be fused, as inquinoline or benzofuran.

“Heteroatom” means any noncarbon atom in a hydrocarbon analog compound.Examples include oxygen, sulfur, nitrogen, phosphorus, arsenic, silicon,selenium, tellurium, tin, and boron.

“Substituted heterocycle” is heterocycle having one or more side chainsformed from noninterfering substituents.

“Noninterfering substituents” are those groups that yield stablecompounds. Suitable noninterfering substituents or radicals include, butare not limited to, halo, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,C₁₋₁₀ alkoxy, C₇₋₁₂ aralkyl, C₇₋₁₂ alkaryl, C₃₋₁₀ cycloalkyl, C₃₋₁₀cycloalkenyl, phenyl, substituted phenyl, toluoyl, xylenyl, biphenyl,C₂₋₁₂ alkoxyalkyl, C₇₋₁₂ alkoxyaryl, C₇₋₁₂ aryloxyalkyl, C₆₋₁₂ oxyaryl,C₁₋₆ alkylsulfinyl, C₁₋₁₀ alkylsulfonyl, —(CH₂)_(m″)—O—(C₁₋₁₀ alkyl)wherein (m″) is from 1 to 8, aryl, substituted aryl, substituted alkoxy,fluoroalkyl, heterocyclic radical, substituted heterocyclic radical,nitroalkyl, —NO₂, —CN, —NRC(O)—(C₁₋₁₀ alkyl), —C(O)—(C₁₋₁₀ alkyl), C₂₋₁₀thioalkyl, —C(O)O—(C₁₋₁₀ alkyl), —OH, —SO₂, ═S, —COOH, —NR, carbonyl,—C(O)—(C₁₋₁₀ alkyl)-CF₃, —C(O)—CF₃, —C(O)NR₂, —(C₁₋₁₀ alkyl)-S—(C₆₋₁₂aryl), —C(O)—(C₆₋₁₂ aryl), —(CH₂)_(m″)—O—(CH₂)_(m″)—O—(C₁₋₁₀ alkyl)wherein each (m″) is independently selected from 1 to 8, —C(O)NR,—C(S)NR, —SO₂NR, —NRC(O)NR, —NRC(S)NR, salts thereof, and the like. EachR as used herein is independently selected from the group consisting ofH, unsubstituted alkyl, substituted alkyl, unsubstituted aryl,substituted aryl, aralkyl, and alkaryl.

The terms “drug,” “biologically active molecule,” “biologically activemoiety,” “biologically active agent,” “active agent,” and the like areused interchangeably herein and mean any substance which can affect anyphysical or biochemical properties of a biological organism, includingbut not limited to viruses, bacteria, fungi, plants, animals, andhumans. In particular, as used herein, biologically active moleculesinclude any substance intended for diagnosis, cure, mitigation,treatment, or prevention of disease in humans or other animals, or tootherwise enhance physical or mental well-being of humans or animals.Examples of biologically active molecules include, but are not limitedto, peptides, proteins, enzymes, small molecule drugs (e.g., nonpeptidicdrugs), dyes, lipids, nucleosides, oligonucleotides, polynucleotides,nucleic acids, cells, viruses, liposomes, microparticles and micelles.Classes of biologically active agents that are suitable for use with theinvention include, but are not limited to, antibiotics, fungicides,anti-viral agents, anti-inflammatory agents, anti-tumor agents,cardiovascular agents, anti-anxiety agents, hormones, growth factors,steroidal agents, and the like.

“Polyolefinic alcohol” refers to a polymer comprising an olefin polymerbackbone, such as polyethylene, having multiple pendant hydroxyl groupsattached to the polymer backbone. An exemplary polyolefinic alcohol ispolyvinyl alcohol.

As used herein, “nonpeptidic” refers to a structure substantially freeof amino acids connected via peptide linkages. Thus, for example, whennonpeptidic is used in reference to a polymer backbone, the polymerbackbone is substantially free of amino acids connected via peptidelinkages. The polymer backbone may, however, include a minor number ofpeptide linkages spaced along the length of the backbone, such as, forexample, no more than about 1 peptide linkage per about 50 monomerunits.

“Polypeptide” or “poly(amino acid)” refers to any molecule comprising aseries of amino acid residues linked through amide linkages along thealpha carbon backbone. Modifications of the peptide side chains may bepresent, along with glycosylations, hydroxylations, and the like.Additionally, other nonpeptidic molecules, including lipids and smalldrug molecules, may be attached to the polypeptide. The polypeptide maycomprise any combination or sequence of amino acid residues.

“Amino acid” refers to organic acids containing both a basic amine groupand an acidic carboxyl group. The term encompasses essential andnonessential amino acids and both naturally occurring and synthetic ormodified amino acids. The most common amino acids are listed herein byeither their full name or by the three letter or single letterabbreviations: Glycine (Gly, G); Alanine (Ala, A); Valine (Val, V);Leucine (Leu, L); Isoleucine (Ile, I); Methionine (Met, M); Proline(Pro, P); Phenylalanine (Phe, F); Tryptophan (Trp, W); Serine (Ser, S);Threonine (Thr, T); Asparagine (Asn, N); Glutamine (Gln, Q); Tyrosine,(Tyr, Y); Cysteine (Cys, C); Lysine (Lys, K); Arginine (Arg, R);Histidine (His, H); Aspartic Acid (Asp, D); and Glutamic acid (Glu, E).Reference to an amino acid is without regard to absolute configurationand the description includes amino acids in either the L or D form.

By “residue” is meant the portion of a molecule remaining after reactionwith one or more molecules. For example, an amino acid residue in apolypeptide chain is the portion of an amino acid remaining afterforming peptide linkages with adjacent amino acid residues.

“Hydrophobic” refers to molecules having a greater solubility in octanolthan in water, typically having a much greater solubility in octanol.Conversely, “hydrophilic” refers to molecules having a greatersolubility in water than in octanol.

“Oligomer” refers to short monomer chains comprising 2 to about 20monomer units, preferably 2 to about 10 monomer units.

“Unimolecular” means the entire molecule is covalently bonded togetherin a single molecular structure, rather than reliant on othernoncovalent bonding or attraction forces as in a traditional micelle.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of adrug, and includes both humans an animals.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

II. THE UNIMOLECULAR MULTI-ARM BLOCK COPOLYMER

The present invention provides a unimolecular multi-arm block copolymerhaving an inner core region defined by a central core molecule havingpolypeptide segments covalently attached thereto and an outerhydrophilic region defined by hydrophilic polymer segments covalentlyattached to each of the polypeptide polymer segments. Thus, each arm ofthe multi-arm structure is a block copolymer comprising an inner (i.e.closer or proximal to the central core molecule) polypeptide polymersegment and an outer (i.e. further or distal from the central coremolecule) hydrophilic polymer segment.

The unimolecular multi-arm block copolymers of the invention areparticularly well suited for encapsulation or entrapment of biologicallyactive molecules within the inner core region. As used herein,“encapsulation” or “entrapment” is intended to refer to the physicalconfinement of a drug molecule within the inner core region of thecopolymer, whether by covalent attachment, charge interaction,metal-acid complex, van der Waals forces, or other attraction or bondingforce.

The unimolecular multi-arm block copolymer typically has a total numberaverage molecular weight of from about 5,000 Da to about 120,000 Da,preferably from about 10,000 Da to about 100,000 Da, and more preferablyfrom about 20,000 Da to about 80,000 Da. An illustration of the generalstructure of the block copolymer of the invention is shown in FIG. 1.

The outer hydrophilic polymer segments are preferably poly(ethyleneglycol), although other hydrophilic polymer segments can also be used.In certain embodiments, a targeting moiety that can direct the copolymerstructure to particular sites within the body is attached to one or moreof the hydrophilic polymer segments for targeted release of an entrappeddrug.

The use of a polypeptide polymer segment as part of the inner coreregion of the unimolecular multi-arm structure provides tremendousflexibility in designing and adjusting the drug delivery properties ofthe multi-arm structure. Interaction between a drug and the core regionof the unimolecular multi-arm structure can greatly affect drug loadingand drug release characteristics. In the present invention, depending onthe structure of the polypeptide polymer segments, the inner core regionof the unimolecular multi-arm structure can be hydrophobic, charged,suitable for covalent attachment to drug molecules, or any combinationthereof.

Hydrophobic Core Region

Polypeptides can exhibit a broad range of hydrophobicity. When ahydrophobic polypeptide is utilized, it is believed that the multi-armblock copolymer acts as a unimolecular micelle in aqueous solution, themicelle structure comprising a central hydrophobic core region boundedby a hydrophilic outer region. As a result, in this embodiment, themulti-arm block copolymers of the invention are capable of increasingthe aqueous solubility of hydrophobic biologically active agents byencapsulating or physically entrapping them within the hydrophobic coreregion of the multi-arm block copolymer structure. The exact structureof the polypeptide, and consequently the hydrophobicity thereof, can beadjusted as necessary to maximize affinity for a particular drugmolecule.

Compared to conventional linear micelle structures, the unimolecularnature of the multi-arm block copolymers of the invention results inless sensitivity to concentration, such that the block copolymers of theinvention are less likely to release the entrapped drug molecules at anundesirably rapid rate. The multi-arm block copolymers of the inventionare covalently bound molecular units rather than aggregates ofindividual molecules. As a result, unintended disassembly of thestructure in vivo due to insufficient concentrations of themicelle-forming materials is avoided with the presently describedmulti-arm block copolymers. Intentional disassembly of the multi-armblock copolymers, however, can occur in vivo by including one or morehydrolytically unstable linkages within the polymer segments. Incontrast to the spontaneous concentration-dependent disassemblyassociated with aggregates of molecules, the multi-arm block copolymersof the invention having one or more hydrolytically unstable linkagesadvantageously disassemble over time in a predictable manner based onthe rate of hydrolysis. Further, since chemical modification of theactive agent is not required to obtain an increase in solubility, thepossibility of the copolymer reducing efficacy of the entrapped drug isgreatly reduced.

Although not wishing to be bound by any particular theory, it isbelieved that the level of hydrophobicity and size of the hydrophobicpolypeptide affect the drug loading and drug release characteristics ofthe multi-arm block copolymer. In general, it is believed that largerhydrophobic polypeptide segments and hydrophobic polypeptide segmentsformed from amino acids having relatively greater degrees ofhydrophobicity will result in higher drug loading and slower drugrelease profiles in solution. Conversely, smaller hydrophobicpolypeptide segments and hydrophobic polypeptide segments formed fromamino acids having relatively lower degrees of hydrophobicity willresult in reduced drug loading and more rapid drug release. Usingroutine experimentation, one of ordinary skill in the art can determinean appropriate polypeptide segment composition and size for any givendrug. For example, a series of drug-containing multi-arm blockcopolymers can be prepared as discussed herein, each having a differencesize of polypeptide segment. The multi-arm block copolymer having thegreatest efficacy upon administration to a patient has an appropriatelysized polypeptide segment. A similar approach can be used to determinean appropriate polypeptide segment content (i.e., the amino acidresidues present in the polypeptide segment). Here, however, a series ofdrug-containing multi-arm block copolymers having different amino acidresidues in the polypeptide segment are tested.

Charged Core Region

In another embodiment, hydrophilic biologically active agents that beara charge in aqueous media can be entrapped within the inner core regionof the unimolecular multi-arm block copolymers of the invention byselecting a polypeptide segment having an opposite charge. The oppositecharges result in attraction between the core region of the multi-armstructure and the drug. For example, if an inner core region with apositive charge is needed to entrap a negatively charge active agent, apolypeptide segment can be formed from amino acid residues that arepositively charged, preferably at or near physiological pH (i.e., pH ofabout 7.4). Lysine (having a pK of about 10.0) and arginine (having a pKof about 12.0) are positively charged at or near physiological pH. Otheramino acids can also be positively charged, depending on the pH of theenvironment. For example, the imidazole ring of histidine can bepositively charged.

If an inner core region with a negative charge is desired for an activeagent bearing a positive charge, a polypeptide segment can be formedfrom amino acid residues that are preferably negatively charged,preferably at or near physiological pH (i.e., pH of about 7.4). Glutamicacid and aspartic acid (both having a pK of about 4.4) are negativelycharged at or near physiological pH. Depending on the pH of theenvironment, other amino acids such as cysteine can also be used toprovide a negative charge.

The use of charge attraction to entrap a biologically active molecule isparticularly advantageous for entrapping DNA, RNA or oligonucleotides.The entrapped drug is released in a sustained and extended manner fromthe charged inner core region. An illustration of a charged-coreembodiment of the block copolymer of the invention is shown in FIG. 2.Areas of the polypeptide segment wherein an amino acid residues bears anegative charge are depicted with a negative (“−”) sign.

Core Regions Suitable for Covalent Attachment to Drugs

In yet another embodiment, the biologically active agent can becovalently attached to the inner core region of the multi-arm blockcopolymer of the invention by covalently attaching the biologicallyactive agent to a pendant functional group spaced within the polypeptidechain. For example, polypeptides formed from a number of different aminoacids, such as aspartic acid and glutamic acid, include pendantcarboxylic acid groups on their side chains. These acid groups canreadily react with, for example, a biologically active agent bearing anamine group, thereby linking the biologically active agent to thepolypeptide chain via an amide linkage. In addition, a polypeptidesegment comprising an amine-containing lysine residue can react with abiologically active agent bearing a carboxylic acid group, therebyforming an amide linkage. An illustration of covalent attachment of drugmolecules to the block copolymer of the invention is shown in FIG. 3.

If desired, the linkage between the polypeptide and the biologicallyactive agent can be a degradable linkage, such as an ester linkage,carbonate linkage, imine linkage, hydrazone linkage, acetal linkage, orortho ester linkage. In this manner, the block copolymer can actessentially as a prodrug, releasing the drug molecules upon hydrolysisof the degradable linkages in solution.

In any of the above embodiments, as compared to linear block copolymers,the multi-arm block copolymers of the invention can better protect thedrug molecules from enzymatic degradation by sheltering the drug withinthe inner core region. Also, in embodiments incorporating a targetingmoiety, the targeting moiety can be used more efficiently as compared tolinear block copolymers. In the present case, a targeting moietyattached to only a few copolymer arms can effectively deliver a numberof drug molecules entrapped within the core region of the multi-armstructure, thereby increasing the delivered drug “payload” relative to atargeted linear polymer having only one or two drug moieties covalentlyattached thereto.

It is believed that the number of arms of the multi-arm block polymerhas an impact on the drug loading and drug release characteristics ofthe copolymer embodiments having a hydrophobic or charged core region.Generally, the presence of fewer copolymer arms results in reduced drugloading and more rapid drug release. The use of a copolymer with a verylarge number of arms can also reduce drug loading because of thesubstantial increase in density and concomitant reduction ininterstitial space within the core region of the copolymer structure.Copolymers with a higher number of arms are, however, less likely tohave drug release characteristics that depend on concentration. In lightof the foregoing, an optimal range for the number of arms of the blockcopolymer can be determined such that both desirable drug loading anddrug release characteristics are obtained for any particular hydrophobicdrug. One of ordinary skill in the art can determine through routineexperimentation what number of arms is appropriate in any given context.For example, one of ordinary skill in the art can create a series ofdifferently numbered arms of drug-containing multi-arm block copolymers,monitor the efficacy of each copolymer in the series upon injection intoa patient, and subsequently identify the appropriate number of arms asbeing associated with the number of arms in the copolymer having thebest efficacy. In most embodiments, however, the number of arms is inthe range of from 3 to about 25, and can therefore be 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. Itis preferred, however, that the number of arms in the multi-arm blockcopolymers described herein is at least 5, more preferably at leastabout 8, and most preferably at least about 10.

The polypeptide and hydrophilic polymer segments are preferably not“hyper-branched” or dendritic in nature, such as the dendrimersdescribed in U.S. Pat. No. 5,830,986, wherein branched compounds areattached in numerous successive layers to a central core. Instead, bothpolymer segments are preferably substantially linear in nature asdepicted in FIG. 1. However, some branching in either polymer segmentmay be present. For example, a branched poly(ethylene glycol) polymercomprising two polymer backbones attached to lysine linker can be usedas the hydrophilic polymer.

Although the specific examples of multi-arm block copolymers in theappended experimental section utilize the same block copolymer structurefor each copolymer arm, it is possible to utilize different copolymerstructures within the same multi-arm structure. In other words, thepresent invention includes embodiments wherein more than onepolypeptide/hydrophilic polymer combination is attached to the same coremolecule.

A. The Central Core

The central core molecule is derived from a molecule that provides anumber of polymer attachment sites equal to the number of desiredcopolymer arms. By “attachment site” is meant a functional group capableof reacting with another molecule, such as an amino acid, to form acovalent linkage. Preferably, the central core molecule is a residue ofa polyamine having at least three termini bearing an amine group. By“polyamine” is meant a branched molecule containing a plurality ofterminal amine groups available as attachment sites for attaching thecopolymer arms to the core molecule. The use of a polyamine core ispreferred because the amine groups of the core readily react with thecarboxylic acid group of an amino acid to form an amide linkage. Coremolecules having other functional groups available for attachment to thecopolymer arms can, however, also be used.

In embodiments utilizing a polyamine core, the number of amine groupswill dictate the number of copolymer arms in the multi-arm structure.Preferably, the polyamine comprises from 3 to about 25 amine groups. Invarious embodiments, the polyamine comprises at least about 5 aminegroups, at least about 8 amine groups, or at least about 10 aminegroups.

The core molecule typically has a total number average molecule weightof from about 250 Da to about 15,000 Da, preferably from about 500 Da toabout 10,000 Da, and more preferably from about 1,000 Da to about 5,000Da. Thus, exemplary total number average molecular weights of the coremolecule include the following: about 250 Da; about 300 Da; about 350Da; about 400 Da; about 450 Da; about 500 Da; about 550 Da; about 600Da; about 650 Da; about 700 Da; about 750 Da; about 800 Da; about 850Da; about 900 Da; about 950 Da; about 1,000 Da; about 1,500 Da; about2,000 Da; about 2,500 Da; about 3,000 Da; about 3,500 Da; about 4,000Da; about 4,500 Da; about 5,000 Da; about 5,500 Da; about 6,000 Da;about 6,500 Da; about 7,000 Da; about 7,500 Da; about 8,000 Da; about8,500 Da; about 9,000 Da; about 9,500 Da; about 10,000 Da; about 10,500Da; about 11,000 Da; about 11,500 Da; about 12,000 Da; about 12,500 Da;about 13,000 Da; about 13,500 Da; about 14,000 Da; about 14,500 Da; andabout 15,000 Da. In the embodiments exemplified in the appendedexperimental section, the molecular weight of the polyamine coremolecule is about 2,000 Da.

In a preferred embodiment, the polyamine core molecule is formed bycovalent attachment of small molecular weight hydrophilic oligomersbearing an amine group to a polyol core. In this embodiment, the centralcore molecule comprises the residue of a polyol having at least threehydroxyl groups available for polymer attachment. A “polyol” is amolecule comprising a plurality of available hydroxyl groups. Dependingon the desired number of copolymer arms, the polyol will typicallycomprise from about 3 to about 25 hydroxyl groups, preferably at least5, more preferably at least about 8, and most preferably at least about10. The polyol may include other protected or unprotected functionalgroups as well without departing from the invention. Although thespacing between hydroxyl groups will vary from polyol to polyol, thereare typically about 1 to about 20 atoms, such as carbon atoms, betweeneach hydroxyl group, preferably 1 to about 5. Preferred polyols includeglycerol, reducing sugars such as sorbitol, pentaerythritol, andglycerol oligomers, such as hexaglycerol. For example, a cyclodextrinsuch as hydroxypropyl-β-cyclodextrin, which has 21 available hydroxylgroups, can form a 21-arm block copolymer. The particular polyol chosenwill depend on the desired number of copolymer arms in the multi-armcopolymer structure wherein the number of hydroxyl groups in the polyolwill correspond to the total number of copolymer arms.

When covalently attaching small molecular weight hydrophilic oligomersto a core molecule, each hydrophilic oligomer should have a molecularweight sufficiently small to avoid significantly affecting the level ofhydrophobicity of the inner core region of the multi-arm structure.Typically, an ethylene glycol oligomer chain having a molecular weightfrom about 88 Da to about 1,000 Da, preferably from about 100 Da toabout 1,000 Da, more preferably from about 100 Da to about 500 Da isused. In one embodiment, the ethylene glycol oligomer has a numberaverage molecular weight of about 200 to about 300 Da.

The oligomer segments can be readily attached to the polyol core byusing an oligomer having a functional group suitable for reaction withthe available hydroxyl groups of the polyol. For example, a bifunctionalethylene glycol oligomer having a mesylate group at one terminus and anamine group at the other terminus can be reacted with a polyol to forman ester linkage between the ethylene glycol oligomer and the polyol.The amine groups of the ethylene glycol oligomer would then be availablefor reaction with an amino acid to form the polypeptide segment of thecopolymer arms. Alternatively, the ethylene glycol oligomer can bedirectly polymerized onto the polyol core as described in, for example,U.S. Pat. No. 6,046,305, followed by derivatizing the terminal group ofthe PEG oligomer to form an amine group.

The general structure of a preferred polyamine central core molecule isshown below.A′(-O-PEG_(o)-NH₂)_(n)  Formula Iwherein:

A′ is a residue of a polyol, such as glycerol, sorbitol,pentaerythritol, glycerol oligomers, or a polyol-containingcyclodextrin, e.g., hydroxypropyl-β-cyclodextrin;

PEG_(o) is a PEG oligomer having a molecular weight of about 100 Da toabout 1,000 Da; and

(n) is 3 to about 25, and is used to represent the number of(—O-PEG_(o)-NH₂) moieties attached to the central core molecule.

Polyamine structures in accordance with Formula I are prepared fromcommercially available multi-arm ethylene glycols available from NOFCorporation (Tokyo, Japan) or can be readily prepared using commerciallyavailable reagents as described above.

Also suitable for use as the central core are branched polyamines suchas those described above but absent an ethylene glycol component.

B. The Polypeptide Segment

The polypeptide should be generally nontoxic and biocompatible, meaningthat the benefits derived from its proposed use in connection withliving tissues (e.g., administration to a patient) outweighs anydeleterious effects as evaluated by a clinician, e.g., a physician. Inpreferred embodiments, the polypeptide segments comprise residues ofamino acids such as glycine, alanine, valine, leucine, isoleucine,methionine, proline, phenylalanine, tryptophan, serine, threonine,asparagine, glutamine, tyrosine, cysteine, lysine, arginine, histidine,aspartic acid, glutamic acid, or combinations thereof. Particularlypreferred amino acids that enhance the hydrophobic nature of thepolypeptide segment are selected from the group consisting of valine,leucine, isoleucine, phenylalanine, tryptophan, tyrosine, methionine,and cysteine. If a charged inner core region is desired, the polypeptidepreferably comprises residues of charged or chargeable amino acids, suchas arginine, lysine, histidine, glutamic acid, aspartic acid, orcombinations thereof.

The polypeptide segment of the block copolymer will typically have anumber average molecular weight of about 100 Da to about 20,000 Da,preferably about 500 Da to about 10,000 Da. For example, polypeptidesegments having a molecular weight of about 100 Da, about 200 Da, about300 Da, about 500 Da, about 800 Da, about 1,000 Da, about 2,000 Da,about 3,000 Da, about 4,000 Da, and about 5,000 Da are useful in thepresent invention.

When a polyamine core is utilized, the polypeptide segment is typicallyattached to the central core molecule by amide linkages. The polypeptideis also preferably attached to the hydrophilic polymer segment by anamide linkage. In preferred embodiments, the polypeptide segment has thefollowing structure:(—C(O)—CHR—NH—)_(m)  Formula IIwherein:

R is hydrogen, alkyl (e.g., C₁₋₆ alkyl) wherein one or more carbon atomsof the alkyl chain can be optionally replaced with a heteroatom (e.g.,O, S or N), or substituted alkyl; and

(m) is about 3 to about 100, preferably about 3 to about 50, and morepreferably about 3 to about 30.

Preferred substituents for the R alkyl chain include aryl, substitutedaryl, heteroaryl, substituted heteroaryl, and functional groups, such asthiol, amine, carboxylic acid, esters of carboxylic acid, and the like.As noted above, pendant functional groups spaced along the polypeptidebackbone can be used to covalently attach biologically active agents tothe polypeptide segment. Exemplary functional groups include hydroxyl,active ester (e.g. N-hydroxysuccinimidyl ester or 1-benzotriazolylester), active carbonate (e.g. N-hydroxysuccinimidyl carbonate and1-benzotriazolyl carbonate), acetal, aldehyde, aldehyde hydrate,alkenyl, acrylate, methacrylate, acrylamide, active sulfone, amine,hydrazide, thiol, carboxylic acid, isocyanate, isothiocyanate,maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide,epoxide, glyoxal, dione, mesylate, tosylate, or tresylate.

The polypeptide segment will typically be enzymatically degradable. Theuse of a degradable polypeptide allows the multi-arm block copolymer todegrade in vivo over time, thus increasing renal clearance of thecopolymer. In addition, the degradation of the polypeptide provides anadditional feature of these polymers, i.e., the ability to control therate of release of the entrapped drug.

C. The Hydrophilic Polymer

The hydrophilic polymer segment can comprise any hydrophilic polymer andthe invention is not limited in this regard. As with the polypeptide,the hydrophilic polymer should generally be nontoxic and biocompatible.Preferably, poly(ethylene glycol) (“PEG”) is used as the hydrophilicpolymer segment. The term PEG includes poly(ethylene glycol) in any ofits linear, branched or multi-arm forms, including alkoxy PEG,bifunctional PEG, forked PEG, branched PEG, pendant PEG, or PEG withdegradable linkages therein, to be more fully described below.

In its simplest form, PEG has the formula—CH₂CH₂O—(CH₂CH₂O)_(n″)—CH₂CH₂—  Formula IIIwherein (n″) is from about 2 to about 4,000, typically about 10 to about4,000, and more typically from about 20 to about 500.

Although the number average molecular weight of the PEG polymer backbonecan vary, PEGs having a number average molecular weight of from about100 Da to about 20,000 Da, preferably about 500 Da to about 10,000 Da,are particularly useful as the hydrophilic polymer segment. For example,PEG polymer segments having a molecular weight of about 100 Da, about200 Da, about 300 Da, about 500 Da, about 800 Da, about 1,000 Da, about2,000 Da, about 3,000 Da, about 4,000 Da, about 5,000 Da, about 6,000Da, about 7,000 Da, about 8,000 Da, about 9,000 Da, about 10,000 Da,about 11,000 Da, about 12,000 Da, about 13,000 Da, about 14,000 Da,about 15,000 Da, about 16,000 Da, about 17,000 Da, about 18,000 Da,about 19,000 Da, and about 20,000 Da are useful in the presentinvention.

In one form useful in the present invention, free or nonbound PEG is alinear polymer terminated at each end with hydroxyl groups:HO—CH₂CH₂O—(CH₂CH₂O)_(n″)—CH₂CH₂—OH  Formula IVwherein the definition of (n″) is the same as that provided with respectto Formula III.

The above polymer, alpha-,omega-dihydroxylpoly(ethylene glycol), can berepresented in brief form as HO-PEG-OH where it is understood that the-PEG- symbol represents a structure of Formula III above.

Another type of PEG useful in the present invention is methoxy-PEG-OH,or mPEG in brief, in which one terminus is the relatively inert methoxygroup, while the other terminus is a hydroxyl group that is subject toready chemical modification. The structure of mPEG is given below.CH₃O—CH₂CH₂O(CH₂CH₂O)_(n″)—CH₂CH₂—OH  Formula Vwherein the definition of (n″) is the same as that provided with respectto Formula III.

Multi-armed or branched PEG molecules, such as those described in U.S.Pat. No. 5,932,462, which is incorporated by reference herein in itsentirety, can also be used as the PEG polymer. For example, thehydrophilic PEG segment can have the structure:

wherein:

poly_(a) and poly_(b) are PEG backbones, such as methoxy poly(ethyleneglycol);

R″ is a nonreactive moiety, such as H, methyl or a PEG backbone; and

P and Q are nonreactive linkages. In a preferred embodiment, thebranched PEG polymer is methoxy poly(ethylene glycol) disubstitutedlysine.

The PEG polymer may alternatively comprise a forked PEG. An example of aforked PEG is represented by PEG-YCHZ₂, where Y is a linking group and Zis an activated terminal group, linked to CH by a chain of atoms ofdefined length. International Application No. PCT/US99/05333, thecontents of which are incorporated by reference herein, disclosesvarious forked PEG structures capable of use in the present invention.The chain of atoms linking the Z functional groups to the branchingcarbon atom serve as a tethering group and may comprise, for example, analkyl chain, ether linkage, ester linkage, amide linkage, or acombination thereof.

The PEG polymer may comprise a pendant PEG molecule having reactivegroups, such as carboxyl, covalently attached along the length of thePEG backbone rather than at the end of the PEG chain. The pendantreactive groups can be attached to the PEG backbone directly or througha linking moiety, such as an alkylene group.

In addition to the above-described forms of PEG, the polymer can also beprepared with one or more weak or degradable linkages in the polymerbackbone, including any of the above described polymers. For example,PEG can be prepared with ester linkages in the polymer backbone that aresubject to hydrolysis. As shown below, this hydrolysis results incleavage of the polymer into fragments of lower molecular weight:-PEG-CO₂-PEG-+H₂O→-PEG-CO₂H+HO-PEG-

Other hydrolytically degradable linkages, useful as a degradable linkagewithin a polymer backbone, include carbonate linkages; imine linkagesresulting, for example, from reaction of an amine and an aldehyde (see,e.g., Ouchi et al. (1997) Polymer Preprints 38(1):582-3, which isincorporated herein by reference.); phosphate ester linkages formed, forexample, by reacting an alcohol with a phosphate group; hydrazonelinkages which are typically formed by reaction between a hydrazide andan aldehyde; acetal linkages that are typically formed by reactionbetween an aldehyde and an alcohol; ortho ester linkages that areformed, for example, by reaction between a formate and an alcohol; andoligonucleotide linkages formed, for example, by reaction between aphosphoramidite group, e.g., at the end of a polymer, and a 5′ hydroxylgroup of an oligonucleotide.

It is understood by those skilled in the art that the term poly(ethyleneglycol) or PEG represents or includes all the above forms of PEG.

Many other polymers are also suitable for the invention. Polymerbackbones that are nonpeptidic and water-soluble, with from 2 to about300 termini, are particularly useful in the invention. Examples ofsuitable polymers include, but are not limited to, other poly(alkyleneglycols), copolymers of ethylene glycol and propylene glycol,poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol),polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), such asdescribed in U.S. Pat. No. 5,629,384, which is incorporated by referenceherein in its entirety, and copolymers, terpolymers, and mixturesthereof. These polymers may be linear, or may be in any of theabove-described forms (e.g., branched, forked, and the like).

Those of ordinary skill in the art will recognize that the foregoinglist of hydrophillic polymer backbones is by no means exhaustive and ismerely illustrative, and that all polymeric materials having thequalities described above are contemplated.

A targeting moiety or drug molecule can optionally be covalentlyattached to the hydrophilic polymer segment. As used herein, “targetingmoiety” includes any chemical moiety capable of binding to, or otherwiseexhibiting an affinity for, a particular receptor, ligand, type oftissue, or component of any of the foregoing. The addition of atargeting moiety to the copolymer structure can direct the copolymer toparticular sites within the body for targeted release of the physicallyentrapped drug. For example, certain moieties are known to exhibit anaffinity for hydroxyapatite surfaces (i.e., calcium phosphate), such asbone. Exemplary hydroxyapatite-targeting moieties include tetracycline,calcein, bisphosphonates, such as4-amino-1-hydroxybutane-1,1-diphosphonic acid, ditetrabutylammonium salt(AHBDP) or derivatives thereof, polyaspartic acid, polyglutamic acid,and aminophosphosugars. Additional targeting moieties include proteins,antibodies, antibody fragments, peptides, carbohydrates, lipids,oligonucleotides, DNA, RNA, or small molecules having a molecular weightless than 2,000 Da. In a preferred embodiment, the targeting moiety ismono-folic acid or anti-EGFr Fab. Folic acid is especially preferred fortargeted delivery of anticancer agents via attachment to the multi-armcopolymer delivery vehicles as described herein. Folic acid, as atargeting agent, is useful for targeting tumors that overexpress folatereceptors. Exemplary tumors falling into this category include ovariancarcinomas, and solid tumors such as head and neck tumors, lung cancersand colorectal cancers. Thus, the multi-arm block copolymers of theinvention, when attached to folic acid, are particularly preferred fordelivery of anticancer agents useful in the prevention or treatment ofany of the aforementioned cancers.

The PEG polymer segment may further include one or more capping groupsor functional groups covalently attached to the PEG molecule, such as ata terminus of the PEG segment distal from the point of attachment to thepolypeptide. The capping group is typically a relatively inert group,such as an alkoxy group (e.g. methoxy or ethoxy) or benzyloxy. In oneembodiment, one or more of the PEG polymer segments bear a functionalgroup capable of reacting with a targeting moiety or drug molecule sothat such molecules can be attached to the PEG polymer. Exemplaryfunctional groups include hydroxyl, active ester (e.g.N-hydroxysuccinimidyl ester or 1-benzotriazolyl ester), active carbonate(e.g. N-hydroxysuccinimidyl carbonate and 1-benzotriazolyl carbonate),acetal (as used herein, the term “acetal” encompasses ketals as well),aldehyde, aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide,active sulfone, amine, hydrazide, thiol, carboxylic acid, isocyanate,isothiocyanate, maleimide, vinylsulfone, dithiopyridine, vinylpyridine,iodoacetamide, epoxide, glyoxal, dione, mesylate, tosylate, andtresylate.

Specific examples of terminal functional groups for the polymerbackbones of the invention include: N-succinimidyl carbonate (see e.g.,U.S. Pat. Nos. 5,281,698, and 5,468,478); amine (see, e.g., Buckmann etal. (1981) Makromol. Chem. 182:1379, and Zalipsky et al. (1983) Eur.Polym. J. 19:1177); hydrazide (see, e.g., Andresz et al. (1978)Makromol. Chem. 179:301); succinimidyl propionate and succinimidylbutanoate (see, e.g., Olson et al. in Poly(ethylene glycol) Chemistry &Biological Applications, pp 170-181, Harris & Zalipsky Eds., ACS,Washington, D.C., 1997, and U.S. Pat. No. 5,672,662); succinimidylsuccinate (see, e.g., Abuchowski et al. (1984) Cancer Biochem. Biophys.7:175, and Joppich et al. (1979) Makromol. Chem. 180:1381); succinimidylester (see, e.g., U.S. Pat. No. 4,670,417); benzotriazole carbonate(see, e.g., U.S. Pat. No. 5,650,234); glycidyl ether (see, e.g., Pithaet al. (1979) Eur. J. Biochem. 94:11, and Elling et al. (1991) Biotech.Appl. Biochem. 13:354); oxycarbonylimidazole (see, e.g., Beauchamp etal. (1983) Anal. Biochem. 131:25, and Tondelli et al. (1985) J.Controlled Release 1:251); p-nitrophenyl carbonate (see, e.g., Veroneseet al. (1985) Appl. Biochem. Biotech. 11:141, and Sartore et al. (1991)Appl. Biochem. Biotech. 27:45); aldehyde (see, e.g., Harris et al.(1984) J. Polym. Sci. Chem. Ed. 22:341, and U.S. Pat. Nos. 5,824,784,and 5,252,714); maleimide (see, e.g., Goodson et al. (1990)Bio/Technology 8:343, Romani et al. (1984) Chemistry of Peptides andProteins 2:29), and Kogan (1992) Synthetic Comm. 22:2417);orthopyridyl-disulfide (see, e.g., Woghiren et al. (1993) Bioconj. Chem.4:314); acrylol (see, e.g., Sawhney et al. (1993) Macromolecules26:581); and vinylsulfone (see, e.g., U.S. Pat. No. 5,900,461). All ofthe above references are incorporated herein by reference.

Exemplary Unimolecular Multi-Arm Block Copolymer Structures

More specific structural embodiments of the block copolymers of theinvention will now be described. The specific structures shown below arepresented as exemplary structures only, and are not intended to limitthe scope of the invention.

In one embodiment, the block copolymer of the invention has thestructure:A(-O—B-L₁-C)_(m′)-(L₂-D-E)_(n′)  Formula VIIwherein:

A is a central core molecule comprising a residue of a polyol, such asglycerol, sorbitol, pentaerythritol, glycerol oligomers, orpolyol-containing cyclodextrin such as hydroxypropyl-β-cyclodextrin;

O is oxygen;

B is a hydrophilic oligomer, such as a PEG oligomer;

C is a polypeptide segment;

D is a hydrophilic polymer segment, such as a PEG polymer;

E is a capping group (e.g., alkoxy) or a functional group (e.g.,hydroxy, active esters, and so forth);

L₁ and L₂ are linkages, such as amide linkages;

(m′) is 3 to about 25 and is used to represent the number of (—O—B-L₁-C)moieties attached to central core molecule;

(n′) is 2 to about 25 and is used to represent the number of (L₂-D-E)moieties attached to the (m′) number of (—O—B-L₁-C) moieties, each(—O—B-L₁-C) moiety bearing a single (L₂-D-E) moiety; and

(n′)≦(m′).

A preferred embodiment comprising a targeting moiety has the structure:(T-D-L₂-C-L₁-B—O-)_(p)A(-O—B-L₁-C-L₂-D-E)_(k)  Formula VIIIwherein:

each A, O, B, C, D, E, L₁ and L₂ is independently as described abovewith respect to Formula VII;

T is a targeting moiety;

(p) is a positive integer of at least 1 or greater and represents thenumber of (T-D-L₂-C-L₁-B—O—) moieties attached to the central coremolecule;

(k) is a positive integer of at least 1 or greater and represents thenumber of (—O—B-L₁-C-L₂-D-E) moieties attached to the central coremolecule; and

the sum of (k) and (p) is from 3 to about 25. In one embodiment, (p) is1 to about 5, preferably 1 to about 3, and the sum of (k) and (p) isabout 6 to about 21, preferably about 8 to about 15.

Formula IX below is an exemplary unimolecular 8-arm polypeptide-PEGblock copolymer made in accordance with the invention.

wherein:

each POLYPEPTIDE is a polypeptide segment, preferably a polypeptideformed from residues of aspartic acid, glutamic acid or lysine; and

each PEG is poly(ethylene glycol), preferably including a terminalcapping or functional group as described above.

Particularly preferred structures for POLYPEPTIDE in Formula IX areshown below:

wherein (m) is as defined above with respect to Formula II.

E. The Biologically Active Agent

The biologically active moiety or drug that is carried within theunimolecular multi-arm block copolymer of the invention may be anybiologically active agent capable of being physically entrapped withinthe block copolymer structure. The entrapped or encapsulated drug may beutilized per se or in the form of a pharmaceutically acceptable salt. Ifused, a salt of the drug compound should be both pharmacologically andpharmaceutically acceptable, but nonpharmaceutically acceptable saltsmay conveniently be used to prepare the free active compound and are notexcluded from the scope of this invention. Pharmacologically andpharmaceutically acceptable salts can be prepared by reaction of thedrug with an organic or inorganic acid, using standard methods detailedin the literature. Examples of useful salts include, but are not limitedto, those prepared from the following acids: hydrochloric, hydrobromic,sulfuric, nitric, phosphoric, maleic, acetic, salicyclic,p-toluenesulfonic, tartaric, citric, methanesulphonic, formic, malonic,succinic, naphthalene-2-sulphonic and benzenesulphonic, and the like.Also, pharmaceutically acceptable salts can be prepared as alkalinemetal or alkaline earth salts, such as sodium, potassium, or calciumsalts of a carboxylic acid group.

Examples of hydrophobic drug molecules that may be encapsulated withinthe multi-arm block copolymer in embodiments comprising a hydrophobiccore region include, but are not limited to, abietic acid, aceglatone,acenaphthene, acenocoumarol, acetohexamide, acetomeroctol, acetoxolone,acetyldigitoxins, acetylene dibromide, acetylene dichloride,acetylsalicylic acid, alantolactone, aldrin, alexitol sodium, allethrin,allylestrenol, allylsulfide, alprazolam, aluminum bis(acetylsalicylate),ambucetamide, aminochlothenoxazin, aminoglutethimide, amyl chloride,androstenediol, anethole trithone, anilazine, anthralin, Antimycin A,aplasmomycin, arsenoacetic acid, asiaticoside, asternizole, aurodox,aurothioglycanide, 8-azaguanine, azobenzene, baicalein, Balsam Peru,Balsam Tolu, barban, baxtrobin, bendazac, bendazol, bendroflumethiazide,benomyl, benzathine, benzestrol, benzodepa, benzoxiquinone,benzphetamine, benzthiazide, benzyl benzoate, benzyl cinnamate,bibrocathol, bifenox, binapacryl, bioresmethrin, bisabolol, bisacodyl,bis(chlorophenoxy)methane, bismuth iodosubgallate, bismuth subgallate,bismuth tannate, Bisphenol A, bithionol, bornyl, bromoisovalerate,bornyl chloride, bornyl isovalerate, bornyl salicylate, brodifacoum,bromethalin, broxyquinoline, bufexamac, butamirate, butethal,buthiobate, butylated hydroxyanisole, butylated hydroxytoluene, calciumiodostearate, calcium saccharate, calcium stearate, capobenic acid,captan, carbamazepine, carbocloral, carbophenothin, carboquone,carotene, carvacrol, cephaeline, cephalin, chaulmoogric acid, chenodiol,chitin, chlordane, chlorfenac, chlorfenethol, chlorothalonil,chlorotrianisene, chlorprothixene, chlorquinaldol, chromonar,cilostazol, cinchonidine, citral, clinofibrate, clofaziminc, clofibrate,cloflucarban, clonitrate, clopidol, clorindione, cloxazolam, coroxon,corticosterone, cournachlor, coumaphos, coumithoate cresyl acetate,crimidine, crufomate, cuprobam, cyamemazine, cyclandelate, cyclarbamatecymarin, cyclosporin A, cypermethril, dapsone, defosfamide,deltamethrin, deoxycorticocosterone acetate, desoximetasone,dextromoramide, diacetazoto, dialifor, diathymosulfone, decapthon,dichlofluani, dichlorophen, dichlorphenamide, dicofol, dicryl,dicumarol, dienestrol, diethylstilbestrol, difenamizole,dihydrocodeinone enol acetate, dihydroergotamine, dihydromorphine,dihydrotachysterol, dimestrol, dimethisterone, dioxathion, diphenane,N-(1,2-diphenylethyl)nicotinamide, 3,4-di-[1-methyl6-nitro-3-indolyl]-1H-pyrrole-2,5-dione (MNIPD), dipyrocetyl,disulfamide, dithianone, doxenitoin, drazoxolon, durapatite, edifenphos,emodin, enfenamic acid, erbon, ergocorninine, erythrityl tetranitrate,erythromycin stearate, estriol, ethaverine, ethisterone, ethylbiscournacetate, ethylhydrocupreine, ethyl menthane carboxamide,eugenol, euprocin, exalamide, febarbamate, fenalamide, fenbendazole,fenipentol, fenitrothion, fenofibrate, fenquizone, fenthion, feprazone,flilpin, filixic acid, floctafenine, fluanisone, flumequine, fluocortinbutyl, fluoxymesterone, fluorothyl, flutazolam, fumagillin,5-furftiryl-5-isopropylbarbituric acid, fusaftmgine, glafenine,glucagon, glutethimide, glybuthiazole, griseofulvin, guaiacol carbonate,guaiacol phosphate, halcinonide, hematoporphyrin, hexachlorophene,hexestrol, hexetidine, hexobarbital, hydrochlorothiazide, hydrocodone,ibuproxam, idebenone, indomethacin, inositol niacinate, iobenzamic acid,iocetamic acid, iodipamide, iomeglamic acid, ipodate, isometheptene,isonoxin, 2-isovalerylindane-1,3-dione, josamycin, 11-ketoprogesterone,laurocapram, 3-O-lauroylpyridoxol diacetate, lidocaine, lindane,linolenic acid, liothyronine, lucensomycin, mancozeb, mandelic acid,isoamyl ester, mazindol, mebendazole, mebhydroline, mebiquine,melarsoprol, melphalan, menadione, menthyl valerate, mephenoxalone,mephentermine, mephenyloin, meprylcaine, mestanolone, mestranol,mesulfen, metergoline, methallatal, methandriol, methaqualone,methylcholanthrene, methylphenidate, 17-methyltestosterone,metipranolol, minaprine, myoral, naftalofos, naftopidil, naphthalene,2-naphthyl lactate, 2-(2-naphthyloxy)ethanol, naphthyl salicylate,naproxen, nealbarbital, nemadectin, niclosamide, nicoclonate,nicomorphine, nifuroquine, nifuroxazide, nitracrine, nitromersol,nogalamycin, nordazepam, norethandrolone, norgestrienone, octaverine,oleandrin, oleic acid, oxazeparn, oxazolam, oxeladin, oxwthazaine,oxycodone, oxymesterone, oxyphenistan acetate, paclitaxel,paraherquamide, parathion, pemoline, pentaerythritol tetranitrate,pentylphenol, perphenazine, phencarbamide, pheniramine,2-phenyl-6-chlorophenol, phenthnethylbarbituric acid, phenyloin,phosalone, O-phthalylsulfathiazole, phylloquinone, picadex, pifamine,piketopfen, piprozolin, pirozadil, pivaloyloxymethyl butyrate,plafibride, plaunotol, polaprezinc, polythiazide, probenecid,progesterone, promegestone, propanidid, propargite, propham, proquazone,protionamide, pyrimethamine, pyrimithate, pyrvinium pamoate, quercetin,quinbolone, quizalofo-ethyl, rafoxanide, rescinnamine, rociverine,ronnel, salen, scarlet red, siccanin, simazine, simetride, simvastatin,sobuzoxane, solan, spironolactone, squalene, stanolone, sucralfate,sulfabenz, sulfaguanole, sulfasalazine, sulfoxide, sulpiride,suxibuzone, talbutal, terguide, testosterone, tetrabromocresol,tetrandrine, thiacetazone, thiocolchicine, thioctic acid, thioquinox,thioridazine, thiram, thymyl N-isoamylcarbamate, tioxidazole, tioxolone,tocopherol, tolciclate, tolnaftate, triclosan, triflusal, triparanol,ursolic acid, valinomycin, verapamil, vinblastine, vitamin A, vitamin D,vitamin E, xenbucin, xylazine, zaltoprofen, and zearalenone.

Examples of charged biologically active agents that can be entrappedwithin multi-arm block copolymer embodiments having a charged inner coreregion include cisplatin, lidocaine and its analogues, tolterodine,mitoxantrone, and imatinib (Gleevec).

In embodiments of the block copolymer of the invention having functionalgroups within either the polypeptide segment or the PEG segmentavailable for covalent attachment to drug molecules, any drug moleculehaving an available functional group capable of reacting with thefunctional group of the block copolymer can be used. Such drug moleculesinclude paclitaxel and its analogues, 5-fluorouracil, etoposide,camptothecin and its analogues, vinorelbine and doxorubicin.

III. PHARMACEUTICAL COMPOSITIONS COMPRISING THE MULTI-ARM BLOCKCOPOLYMER

In another aspect, the invention provides pharmaceutical formulations orcompositions, both for veterinary and for human medical use, comprisinga multi-arm block copolymer as described above and at least onebiologically active agent entrapped within the multi-arm blockcopolymer, preferably within the inner core region defined by the coremolecule and the polypeptide segments. As noted previously,incorporation of a hydrophobic drug into a block copolymer structurehaving a hydrophobic core provides the ability to solubilize thehydrophobic drug, which can enhance the circulating residence time ofthe drug upon administration to a patient.

The pharmaceutical formulation can include one or more pharmaceuticallyacceptable carriers, and optionally any other therapeutic ingredients,stabilizers, or the like. The carrier(s) must be pharmaceuticallyacceptable in the sense of being compatible with the other ingredientsof the formulation and not unduly deleterious to the recipient thereof.The compositions of the invention may also include polymericexcipients/additives or carriers, e.g., polyvinylpyrrolidones,derivatized celluloses such as hydroxymethylcellulose,hydroxyethylcellulose, and hydroxypropylmethylcellulose, Ficolls (apolymeric sugar), hydroxyethylstarch (HES), dextrates (e.g.,cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin andsulfobutylether-β-cyclodextrin), polyethylene glycols, and pectin. Thecompositions may further include diluents, buffers, binders,disintegrants, thickeners, lubricants, preservatives (includingantioxidants), flavoring agents, taste-masking agents, inorganic salts(e.g., sodium chloride), antimicrobial agents (e.g., benzalkoniumchloride), sweeteners, antistatic agents, surfactants (e.g.,polysorbates such as “TWEEN 20” and “TWEEN 80,” and pluronics such asF68 and F88, available from BASF), sorbitan esters, lipids (e.g.,phospholipids such as lecithin and other phosphatidylcholines,phosphatidylethanolamines, fatty acids and fatty esters, steroids [e.g.,cholesterol]), and chelating agents (e.g., EDTA, zinc and other suchsuitable cations). Other pharmaceutical excipients and/or additivessuitable for use in the compositions according to the invention arelisted in “Remington: The Science & Practice of Pharmacy,” 19^(th) ed.,Williams & Williams, (1995), the “Physician's Desk Reference,” 52^(nd)ed., Medical Economics, Montvale, N.J. (1998), and in “Handbook ofPharmaceutical Excipients,” 3^(rd) Ed., Ed. A. H. Kibbe, PharmaceuticalPress, 2000.

The block copolymers of the invention may be formulated in compositionsincluding those suitable for oral, buccal, rectal, topical, nasal,ophthalmic, or parenteral (including intraperitoneal injection,intravenous injection, subcutaneous injection, and intramuscularinjection) administration. The block copolymers can also be used informulations suitable for inhalation. The compositions can convenientlybe presented in unit dosage form and can be prepared by any of themethods well known in the art of pharmacy. All methods include the stepof bringing the block copolymer with drug entrapped therein intoassociation with a carrier. In general, the compositions are prepared bybringing the block copolymer/drug formulation into association with aliquid carrier to form a solution or a suspension, or alternatively,bringing the block copolymer/drug formulation into association withformulation components suitable for forming a solid, optionally aparticulate product, and optionally shaping or compressing the productinto a shaped or compressed delivery form. Solid formulations of theinvention, when particulate, will typically comprise particles withsizes ranging from about 1 nanometer to about 500 microns. In general,for solid formulations intended for intravenous administration,particles will typically range from about 1 nm to about 10 microns indiameter. Generally, particles intended for inhalation will typicallyhave a diameter of from about 0.1 microns to 10 microns, preferably fromabout 1 micron to about 5 microns.

The amount of the biologically active agent or drug in the formulationwill vary depending upon the specific drug employed, its molecularweight, and other factors such as dosage form, target patientpopulation, and other considerations, and will generally be readilydetermined by one skilled in the art. The amount of biologically activeagent in the copolymer formulation will be that amount necessary todeliver a therapeutically effective amount of the drug to a patient inneed thereof to achieve at least one of the therapeutic effectsassociated with the drug. In practice, this will vary depending upon theparticular drug, its activity, the severity of the condition to betreated, the patient population, the stability of the formulation, andthe like. Compositions will generally contain anywhere from about 1% byweight to about 30% by weight drug, typically from about 2% to about 20%by weight drug, and more typically from about 3% to about 15% by weightdrug, and will also depend upon the relative amounts ofexcipients/additives contained in the composition. More specifically,the composition will typically contain at least about one of thefollowing percentages of the entrapped drug: 0.5%, 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, or more by weight.

IV. METHODS OF MAKING THE BLOCK COPOLYMER

The multi-arm block copolymers of the invention can be prepared bycovalently attaching a preformed polypeptide segment to the coremolecule followed by covalently attaching a preformed hydrophilicpolymer segment to the terminus of the polypeptide segment. The linkagesbetween the two polymer segments and between the polypeptide segment andthe core molecule will depend on the functional groups employed.Typically, the linkages will be amide linkages. However, other types oflinkages, such as carbamates, esters, carbonates, and acetals could alsobe used without departing from the invention.

Alternatively, one or more of the polypeptide or hydrophilic polymersegments can be prepared by directly polymerizing monomer units of thepolymer using, for example, a ring-opening polymerization technique. Forexample, in order to attach a polypeptide to a polyamine core molecule,an N-carboxyanhydride of an amino acid can be formed and directlypolymerized onto the polyamine core by ring-opening polymerization in asuitable solvent. Suitable solvents include dimethylformamide (DMF),tetrahydrofuran (THF), dioxane and the like. Exemplary methods forforming an N-carboxyanhydride of an amino acid are illustrated inExamples 1-3. Exemplary ring-opening polymerization techniques forattaching a polypeptide to a polyamine core are illustrated in Examples4-5. Amino acid reagents, including amino acids having protectedβ-carboxylic acid groups, are commercially available from Sigma-AldrichCorporation (St. Louis, Mo.).

In a second step, the product of the first reaction is reacted withmonomer units of ethylene oxide in the presence of a base, such aspotassium naphthalenide, sodium hydride, sodium or potassium alkoxides,or other strong bases, to attach a poly(ethylene glycol) segment to thepolypeptide segment. For the second step, solvents such astetrahydrofuran, dioxane, or toluene can be used.

A combination of the above methods can also be used to form the blockcopolymer of the invention. For example, a ring-opening polymerizationcan be used to form the polypeptide segment followed by covalentattachment of a preformed PEG polymer, as exemplified in Example 6.Typically, if the amino acid residues have pendant functional groupsalong the alpha carbon chain of the polypeptide segment, the functionalgroups are maintained in protected form while the polypeptide isattached to the central core molecule and while the hydrophilic polymeris attached to the terminus of the polypeptide chain. Thereafter, anypendant functional groups can be deprotected and used to couple thepolypeptide to biologically active agents (see Examples 7-8). As shownin Example 7, a benzyl protecting group can be removed from asparticacid residues to provide free carboxylic acid groups along thepolypeptide chain. The carboxylic acid groups can then be reacted with abiologically active agent, such as the 5-fluorouracil exemplified inExample 8, to form a covalent linkage between the polypeptide segmentand the biologically active agent.

V. METHODS OF LOADING THE DRUG INTO THE MULTI-ARM BLOCK COPOLYMER

There are several methods for entrapping a biologically active agent ordrug within the inner core region of the block copolymers of theinvention. Obviously, as described above, the drug can be entrapped bycovalent attachment of the drug to the polypeptide or PEG chain.

For embodiments relying on other attraction or bonding forces for drugloading, such as hydrophobicity or differences in electrical charge,there are several methods for entrapping a drug. For loading of a drugbearing a charge or a drug containing a metal such as platinum, thecopolymer and the drug can simply be mixed in aqueous solution toencourage charge attraction or metal-acid complexing between the drugand the polypeptide segment of the copolymer.

There are three general methods for loading a hydrophobic drug. In afirst method, the drug and the copolymer are co-dissolved in an organicsolvent and then dried to form a solid product. The solid product isredissolved in aqueous solution and filtered to remove insolubleparticles prior to use. In a second method, the drug is suspended in anaqueous solution of the copolymer and subjected to ultrasonication forseveral hours in order to intimately contact the drug molecules and thehydrophobic cores of the copolymer structures. The solution is thenfiltered to remove insoluble particles. In a third method, the drug andthe polymer are mixed in solid form and heated to about 60° C. to form amelt. The melt is stirred for several hours to encourage intimate mixingof the drug and copolymer. After cooling to room temperature, theformulation is ready for immediate use, further processing, or storage.

VI. METHOD OF USING THE MULTI-ARM BLOCK COPOLYMERS

As noted above, the multi-arm block copolymers of the invention can beused to solubilize hydrophobic drug molecules in aqueous solution or toentrap and protect charged drug molecules or any drug molecule capableof covalently attaching to the polypeptide or PEG chains. As a result,the copolymer structures of the invention can be used as drug deliveryvehicles by entrapping a variety of drugs within the copolymerstructure, particularly within the inner core region of the copolymer,and administering a therapeutically effective amount of the multi-armblock copolymer with the biologically active agent entrapped therein toa mammal.

The block copolymers of the invention can be used as drug deliveryvehicles for any condition responsive to a drug molecule capable ofentrapment within the copolymer structure. Thus, the block copolymers ofthe invention can be used in pharmaceutical formulations useful fortreating any condition responsive to the entrapped drug in mammals,including humans. A preferred condition for treatment is cancer. Themethod of treatment comprises administering to the mammal atherapeutically effective amount of a composition or formulationcontaining the multi-arm block copolymer with a drug entrapped therein.The therapeutically effective dosage amount of any specific formulationwill vary somewhat from drug to drug, patient to patient, and willdepend upon factors such as the condition of the patient, the loadingcapacity of the block copolymer, and the route of delivery. As a generalproposition, a dosage from about 0.5 to about 20 mg/kg body weight,preferably from about 1.0 to about 5.0 mg/kg, will have therapeuticefficacy. When administered conjointly with other pharmaceuticallyactive agents, even less of the block copolymer/drug composition may betherapeutically effective.

The block copolymer/drug composition may be administered once or severaltimes a day. The duration of the treatment may be once per day for aperiod of from two to three weeks and may continue for a period ofmonths or even years. The daily dose can be administered either by asingle dose in the form of an individual dosage unit or several smallerdosage units or by multiple administration of subdivided dosages atcertain intervals. Possible routes of delivery include buccally,subcutaneously, transdermally, intramuscularly, intravenously, orally,and by inhalation.

VII. EXPERIMENTAL

The following examples are given to illustrate the invention, but shouldnot be considered in limitation of the invention. Examples 1-3illustrate a method of forming an N-carboxyanhydride of an amino acidhaving a benzyl-protected carboxylic acid group. Examples 4-5 illustratea ring-opening polymerization technique for attaching a polypeptidechain to a polyamine core molecule. Example 6 illustrates a method ofattaching a PEG polymer segment to each polypeptide chain to form blockcopolymer arms attached to a central polyamine core molecule. Example 7illustrates a technique for deprotecting the pendant carboxylic acidgroups spaced along the polypeptide segment of the block copolymer arms.Example 8 illustrates a method of conjugating a biologically activeagent to the pendant carboxylic acid groups of the polypeptide segmentof the block copolymer arms. Example 9 illustrates a method of forming ametal-acid complex between a metal-containing drug, cisplatin, and thecarboxylic acid groups of a poly(aspartic acid) segment. Example 10illustrates the release rate of the biologically active agent from theconjugate formed in Example 8.

Unless otherwise indicated, all PEG reagents are available fromShearwater Corporation of Huntsville, Ala. All NMR data was generated bya 300 MHz NMR spectrometer manufactured by Bruker.

Example 1 Synthesis of N-Carboxyanhydride (NCA) of L-Aspartic Acidβ-Benzyl Ester

L-aspartic acid β-benzyl ester (Sigma-Aldrich) was suspended in 225 mlof tetrahydrofuran (THF). To the suspension was added approximately 13 gof bis(trichloromethyl) carbonate dissolved in 25 ml of THF, and themixture stirred at 50° C. until a solution was obtained. The solvent wasremoved under vacuum. THF was added gradually to the solid residue at65° C. until the material was completely dissolved. Hexane was added andthe solution was gradually cooled to −15° C. The resulting powder wasfiltered and the product was recrystallized twice and dried at roomtemperature in vacuo. ¹H NMR (DMSO-d₆): δ 2.98 (dxd, —CHCH₂ COO—), 4.69(t, —CHCH₂COO—), 5.13 (s, CH ₂—C₆H₅—), 7.35 (m, aromatic H).

Example 2 Synthesis of N-Carboxyanhydride (NCA) of L-Glutamic Acidγ-Benzyl Ester

25 grams of L-glutamic acid γ-benzyl ester (Sigma-Aldrich) was suspendedin 250 ml of THF. To the suspension was added approximately 12.3 g ofbis(trichloromethyl) carbonate dissolved in 25 ml of THF. The mixturewas stirred at 50° C. until a transparent solution was obtained and thesolvent was removed under vacuum. The solid residue was recrystallizedtwice from a mixture of THF/hexane and the product dried at roomtemperature in vacuo. ¹H NMR (DMSO-d₆): δ 1.94 (m, —CHCH₂ CH₂COO—), 2.07(m, —CHCH₂CH₂ COO—), 4.45 (t, —CHCH₂CH₂COO—), 5.1 (s, CH ₂—C₆H₅—), 7.35(m, aromatic H).

Example 3 Synthesis of N-Carboxyanhydride of N-ε-Cbz-Lysine

N-ε-Benzyloxycarbonyl-lysine (“N-ε-Cbz-lysine,” 50 g, Sigma-Aldrich) wassuspended in 500 ml of THF. To the suspension was added 21.2 g ofbis(trichloromethyl) carbonate dissolved in 50 ml of THF. The reactionmixture was heated to 50° C. while stirring. After the reaction mixturebecame transparent (about 15 to 45 minutes), the solution was stirred at50° C. for another hour. The solution was cooled to room temperature,filtered and the filtrate added to 1,500 ml of hexane. The hexanesolution was cooled at −20° C. for 2-3 hours and the resultingprecipitate was collected by filtration and further purified byrecrystallization from THF/hexane. The product was dried under vacuum.¹H NMR (DMSO-d₆): δ 1.33 (m, —CHCH₂ CH₂ CH₂ CH₂COO—), 1.68 (m,CHCH₂CH₂CH₂CH₂ COO—), 4.42 (t, —CHCH₂CH₂—), 5.0 (s, CH ₂—C₆H₅—), 7.31(m, aromatic H).

Example 4 Synthesis of 8-arm Poly(benzyl aspartate)

8-arm PEG₂₅₀-amine (0.5 g, total MW 2,000, NOF Corporation, Tokyo,Japan) was dried under vacuum at 60° C. for 2 hours and then dissolvedin 10 ml of anhydrous dimethylformamide (“DMF”). To the solution wasadded a solution of N-carboxyanhydride of benzyl aspartate (10 g) in 10ml of DMF. The mixture was stirred at 40° C. overnight under N₂. Themixture was filtered and the filtrate was added to 200 ml of ether. Theprecipitate was collected by filtration and dried under vacuum. ¹H NMR(DMSO-d₆): δ 3.5 (m, PEG), δ 2.7 (dxd, —CHCH₂ COO—), 4.60 (t,—CHCH₂COO—), 5.10 (s, CH ₂—C₆H₅—), 7.3 (m, aromatic H).

Example 5 Synthesis of 8-arm Poly(benzyl glutamate)

8-arm PEG₂₅₀-amine (0.2 g, total MW 2,000, NOF Corporation, Tokyo,Japan) was azeotropically dried with 200 ml of CHCl₃ by distilling offall the solvent under vacuum. The solid residue was then dissolved in 10ml of anhydrous DMF. To the solution was added a solution ofN-carboxyanhydride of benzyl aspartate (5.56 g) in 20 ml of DMF. Themixture was stirred at 40° C. overnight under N₂. The product wasprecipitated with 200 ml of ether. The precipitate was collected byfiltration, re-precipitated with DMF/ether, and dried under vacuum. ¹HNMR (DMSO-d₆): δ 3.5 (m, PEG), 2.0 (m, —CHCH₂ CH₂ COO—), 4.45 (t,—CHCH₂CH₂COO—), 5.1 (s, CH ₂—C₆H₅—), 7.35 (m, aromatic H).

Example 6 Synthesis of 8-arm Poly(benzyl aspartate)-PEG₅₀₀₀

8-arm poly(benzyl aspartate) (3 g, from Example 4), mPEG₅₀₀₀-CM (3 g, MW5,000 Da), dicyclohexylcarbodiimide (“DCC,” 1.3 g), N,N-dimethylaminopyridine (“DMAP,” 0.3 g), and 1-hydroxybenzotriazole (“HOBT,” 0.3 g)were dissolved in 30 ml of anhydrous chloroform. To the solution wasadded freshly distilled triethylamine (“TEA,” 1 ml) and the reaction wasstirred overnight. The solvent was removed under vacuum. To the residuewas added 60 ml of isopropyl alcohol (“IPA”) with vigorous stirring. Thesolid was collected by filtration and washed with ether twice. Theproduct was dried under vacuum. ¹H NMR (DMSO-d₆): δ 3.5 (m, PEG), δ 2.7(dxd, —CHCH₂ COO—), 4.60 (t, —CHCH₂COO—), 5.10 (s, CH ₂—C₆H₅—), 7.3 (m,aromatic H).

Example 7 Preparation of 8-arm Poly(aspartic acid)-PEG₅₀₀₀

The 8-arm poly(benzyl aspartate)-PEG₅₀₀₀ (4.5 g, MW 5,000 Da, fromExample 6) was dissolved in 0.1 N sodium hydroxide solution and thesolution was stirred at room temperature for one hour. After adjustmentof pH to 2-3, the solution was dialyzed against water overnight toremove salt, ultrafiltered with a MWCO 50,000 membrane to removeunreacted PEG, washed with water once and then lyophilized. ¹H NMR(D₂O-d₂): δ 3.5 (m, PEG), 2.6 (b, —CHCH₂ COO—), 4.36 (b, —CHCH₂COO—).

Example 8 Conjugation of 5-Fluorouracil (5FU) to 8-arm Poly(asparticacid)-PEG₅₀₀₀

5-fluorouracil (“5-FU,” 1.51 g, Aldrich) was dissolved in 10 ml offormalin (˜40% formaldehyde) and stirred for 1 hour at 60° C. Thesolvent was removed under vacuum for 1 week. To the residue was added 10ml of distilled DMF and the solution was stored at −15° C. until used.

8-arm poly(aspartic acid)-PEG₅₀₀₀ (0.44 g, from Example 7) wasvacuum-dried at 50° C. overnight and 20 ml of distilled chloroform wasadded. Dicyclohexylcarbodiimide 1.98 g, 4-dimethylaminopyridine 0.276 g,and 1-hydroxybenzotriazole 0.153 g are vacuum-dried at room temperatureovernight in a round-bottomed flask, and 10 ml of distilled DMF wasadded. Both solutions were kept at −15° C. until used. 5FU solution (3ml, 3.46 mmol) was added to the 8-arm poly(aspartic acid)-PEG₅₀₀₀solution followed by the addition of carbodiimide solution and themixture was stirred at −15° C. for 5 days. The sample was precipitatedin isopropanol. After filtration, the sample was freeze-dried inbenzene. ¹H NMR (DMSO-d₆): δ 3.5 (m, PEG), 2.6 (b, —CHCH₂ COO—), 4.4 (b,—CHCH₂COO—), 6.67 (s, —NCH₂ O—), 8.12 (s, CH in fluorouracil).

Example 9 Incorporation of cis-Diamminodichloro Platinum into 8-armPoly(aspartic acid)-PEG₅₀₀₀

cis-Diamminodichloro platinum (0.012 g, Aldrich) was dissolved in 20 mlof water. 8-arm poly(aspartic acid)-PEG₅₀₀₀ (0.056 g, from Example 7)was dissolved in 20 ml of water. The solution was diluted to a finalconcentration of aspartate group of 10 mM. The cis-diamminodichloroplatinum solution was added to the copolymer solution to form threeformulations of different concentration ratios of platinum to aspartate.The three formulations prepared had platinum to aspartate ratios of 1:1(designated as “1:1” in FIG. 4), 1:1.5 (designated as “1:1.5” in FIG.4), and 1:2 (designated as “1:2” in FIG. 4). The change in UV absorbanceat 249 nm versus time was plotted for each formulation and is shown inFIG. 4. As shown in FIG. 4, the absorbance at 249 nm was increased withincubation time at room temperature, indicating that thecis-diamminodichloro platinum complexed with poly(aspartic acid)segments in 8-arm poly(aspartic acid)-PEG₅₀₀₀.

Example 10 Hydrolysis Study of Conjugate of 5-fluorouracil (5FU) and8-arm Poly(aspartic acid)-PEG₅₀₀₀

The conjugate of 5-fluorouracil (“5-FU”) and 8-arm poly(asparticacid)-PEG₅₀₀₀ (from Example 8) was dissolved in phosphate buffersolutions (0.1M, pH 7.4). The solution was stored at room temperatureand 37° C. At timed intervals, the solution was analyzed by HPLC(Ultrahydrogel 250, Waters). The time (t_(1/2)) at which 50% of 5-FU wasreleased was 58 hours at room temperature and 13 hours at 37° C.

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains havingthe benefit of the teachings presented in the foregoing descriptions andthe associated tables. Therefore, it is to be understood that theinvention is not to be limited to the specific embodiments disclosed andthat modifications and other embodiments are intended to be includedwithin the scope of the appended claims. Although specific terms areemployed herein, they are used in a generic and descriptive sense onlyand not for purposes of limitation.

1. A method comprising administering to a mammal a pharmaceuticalcomposition, the pharmaceutical comprising: a unimolecular multi-armblock copolymer having the structure:A(-O—B-L₁C)_(m′)-(L₂-D-E)_(n′) wherein: A is a central core moleculecomprising a residue of a polyol selected from the group consisting ofglycerol, a reducing sugar, pentaerythritol,hydroxypropyl-β-cyclodextrin and a glycerol oligomer, O is oxygen, B isa hydrophilic ethylene glycol oligomer chain having a molecular weightof about 100 Da to about 5,000 Da, C is a polypeptide segment, D is ahydrophilic polymer segment selected from the group consisting ofpoly(alkylene glycols), copolymers of ethylene glycol and propyleneglycol, poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol),polyphosphazene, polyoxazoline and poly(N-acryloylmorpholine), E is acapping group or a functional group selected from the group consistingof alkoxy, hydroxyl, active ester, active carbonate, acetal, aldehyde,aldehyde hydrate, alkyl or aryl sulfonate, halide, disulfide, alkenyl,acrylate, methacrylate, acrylamide, active sulfone, amine, hydrazide,thiol, carboxylic acid, isocyanate, isothiocyanate, maleimide,vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide,glyoxal, dione, mesylate, tosylate and tresylate, L₁ is a linkagebetween B and C formed from a chemical reaction, L₂ is a linkage betweenC and D formed from a chemical reaction, (m′) is 3 to about 25, (n′) is2 to about 25, and (n′)≦(m′), wherein the unimolecular multi-arm blockcopolymer has a total molecular weight of about 10,000 Da to about200,000 Da; and, a biologically active agent entrapped within the innerregion of the unimolecular multi-arm block copolymer.
 2. The method ofclaim 1, wherein the polypeptide segment comprises one or more chargedamino acid residues, and wherein the biologically active agent entrappedwithin the inner core region bears a charge opposite of the charge ofthe one or more charged amino acid residues.
 3. The method of claim 1,wherein the biologically active agent is covalently attached to an aminoacid residue of the polypeptide segment.
 4. The method of claim 1,wherein both the inner core region and the biologically active agent arehydrophobic.
 5. The method of claim 1, wherein the biologically activeagent is 5-fluorouracil.
 6. The method of claim 1, wherein thebiologically active agent is cis-diamminochloro platinum.
 7. The methodof claim 1, wherein the biologically active agent is indomethacin. 8.The method of claim 1, wherein the biologically active agent ispaclitaxel.